CN1264741A - Human adenosine-diphosphate ribosyglucose hydrolase protein and its coding sequence - Google Patents

Abstract

The present invention provides a novel human hARGHase protein expressed in human liver cancer tissue and its coding sequence. The present invention also provides a method for preparing the protein and nucleic acid sequence, and a method for detecting human hARGHase nucleic acid sequence and polypeptide in a sample. .

Description

A new human adenosyl diphosphate ribosyl glucohydrolase protein and its coding sequence

The invention relates to the fields of molecular biology, enzymology, physiology, genetic engineering and the like. Specifically, the present invention relates to a hARGHase protein expressed in human liver cancer tissue and its nucleic acid sequence. The present invention also relates to the preparation method and use of the protein and nucleic acid sequence.

Adenosyl diphosphate-ribosylglucose hydrolase (ADP-ribosylglycohydrolase, ARGHase) participates in the metabolism of organisms, and its main function is to hydrolyze ADP-coupled ribosylglucose. (Nature 392, 353-358 (1998)).

We cloned the human homologous gene hARGHase of this enzyme in human liver cancer tissue, and the homology study showed that hARGHase has the same or similar function as this enzyme.

Before the publication of the present invention, there has not been any published or reported human hARGHase protein sequence and nucleic acid sequence encoding this protein.

The first object of the present invention is to provide a new human gene hARGHase (Genbank Accession No.AF212236), which is a human hARGHase protein gene.

The second object of the present invention is to provide a new human protein hARGHase.

The third object of the present invention is to provide a method for producing the above-mentioned novel human hARGHase protein and nucleic acid sequence using recombinant technology.

The present invention also provides the application of the human hARGHase protein polypeptide and coding sequence.

In one aspect of the present invention, a kind of isolated DNA molecule is provided, and this molecule comprises: the nucleotide sequence of the polypeptide that coding has human hARGHase protein activity, and described nucleotide sequence and SEQ ID NO.6 are obtained from The nucleotide sequence of the nucleotide 51-1142 DNA molecule has at least 70% homology; or the nucleotide sequence can be obtained from the nucleotide sequence of SEQ ID NO.6 under moderately stringent conditions The nucleotide sequence at position 51-1142 hybridizes. Preferably, the sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.7. More preferably, said sequence has the nucleotide sequence from nucleotide 51-1142 in SEQ ID NO.6.

In another aspect of the present invention, an isolated human hARGHase protein polypeptide is provided, which includes: a polypeptide having the amino acid sequence of SEQ ID NO.7, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof things. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.7.

In another aspect of the present invention, a vector comprising the above-mentioned DNA molecule is also provided.

In another aspect of the present invention, a host cell transformed with the above vector is also provided. In one example the host cell is E. coli; in another example, the host cell is a eukaryotic cell.

In another aspect of the present invention, there is also provided a method for producing a polypeptide having human hARGHase protein activity, the steps of which are as follows:

(1) The nucleotide sequence encoding the polypeptide having human hARGHase protein activity is operably connected to the expression control sequence to form a human hARGHase protein expression vector, and the nucleotide sequence and SEQ ID NO.6 are derived from nucleosides The nucleotide sequence at positions 51-1142 of acid has at least 70% homology;

(2) transferring the expression vector in step (1) into a host cell to form a recombinant cell of human hARGHase protein;

(3) culturing the recombinant cells in step (2) under conditions suitable for expressing the human hARGHase protein polypeptide;

(4) Isolating a polypeptide having human hARGHase protein activity.

Preferably, the nucleic acid sequence used in the method has the sequence of positions 51-1142 in SEQ ID NO.6.

The invention also provides an antibody specifically binding to the hARGHase protein polypeptide.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state, and also means that the DNA or fragment has been Separated from the components that naturally accompany nucleic acids and that have been separated from the proteins that accompany them in cells.

In the present invention, the term "human hARGHase protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human hARGHase protein activity, such as the 51-1142 nucleotide sequence in SEQ ID NO.6 and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 51-1142 nucleotides of the coding frame of the sequence of SEQ ID NO.6 . Due to the degeneracy of codons, a degenerate sequence with as low as about 70% homology with the 51-1142 nucleotide sequence in SEQ ID NO.6 can also encode the sequence described in SEQ ID NO.7 . The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 51 to 1142 in SEQ ID NO.6 under moderately stringent conditions, preferably under highly stringent conditions. The term also includes at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 70% homology with the nucleotide sequence from nucleotide 51-1142 in SEQ ID NO.6 At least 95% nucleotide sequence.

The term also includes variants of the open reading frame sequence in SEQ ID NO. 6 that encode a protein that has the same function as native human hARGHase. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total substance of the sample. or wet weight). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its native state.

In the present invention, the term "human hARGHase protein or polypeptide" refers to a polypeptide having the sequence of SEQ ID NO.7 of human hARGHase protein activity. The term also includes variations of the sequence of SEQ ID NO. 7 that have the same function as natural human hARGHase. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the human hARGHase protein.

Variant forms of the human hARGHase polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA that can hybridize with human hARGHase DNA under high or low stringent conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human hARGHase polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human hARGHase polypeptide or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human hARGHase polypeptides. Usually, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human hARGHase polypeptide sequence. 100 consecutive amino acids.

In the present invention, "human hARGHase conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO.7, at most 10, preferably at most 8, more preferably at most 5 amino acids are similar or similar in nature Amino acids are substituted to form polypeptides. These conservative variant polypeptides are preferably produced by substitutions according to Table 1. Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The invention also includes analogs of human hARGHase protein or polypeptide. The difference between these analogs and the natural human hARGHase polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the human hARGHase polypeptide of the present invention, the human hARGHase coding sequence can be operably linked to the expression control sequence, thereby forming a human hARGHase protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, such as CHO cells, COS cells and the like.

The invention also provides antibodies specifically binding to human hARGHase, including polyclonal antibodies and monoclonal antibodies.

In the present invention, a series of methods known in the art can be used to prepare antibodies specific for human hARGHase. For example, the purified human hARGHase gene product or its antigenic fragments are injected into animals to produce polyclonal antibodies. Likewise, cells expressing human hARGHase or its antigenic fragments can also be used to immunize animals to produce antibodies. Antibodies produced according to the present invention may also be monoclonal antibodies, which can be produced using hybridoma technology (e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6 : 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976). The antibody of the present invention includes an antibody that can inhibit the function of human hARGHase, and can also be an antibody that does not affect the function of human hARGHase. Each type of antibody can be produced by immunizing the fragment or functional domain of the human hARGHase gene product, and the human hARGHase gene product and its fragment can be produced by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind unmodified forms of the human hARGHase gene product can be obtained by immunizing animals with the gene product produced in prokaryotic cells such as E. coli. Antibodies that bind to post-translationally modified forms, such as glycosylated or phosphorylated proteins or polypeptides, can be obtained by immunizing animals with gene products produced in eukaryotic cells, such as yeast or insect cells.

The human hARGHase antibody of the present invention can be used to identify cells expressing human hARGHase protein or polypeptide, such as Jurkat T cells. For example, a human hARGHase-specific antibody can be labeled with a detectable molecule such as fluorescein isothiocyanate (FITC), and then the human hARGHase-specific antibody is contacted with the cell sample, and then detected by fluorescence microscopy or flow cytometry. Cells bound by human hARGHase-specific antibody.

In addition to detecting human hARGHase on the cell surface, the protein can also be analyzed by Western blotting. Cell lysates can be extracted from cultured cells or tissue samples taken from patients such as liver cancer tissue, and dissolved in a lysis buffer containing a detergent. Cell extracts were then separated by SDS-polyacrylamide gel electrophoresis (along with purified human hARGHase polypeptide as a positive control), followed by transfer to nitrocellulose by electrophoretic hybridization. For immunodetection of human hARGHase polypeptides by Western blot, typical antibody binding detection methods such as autoradiography or alkaline phosphatase detection methods can be used. Inoculation serum or irrelevant monoclonal antibodies can be used as controls for non-specific reactions.

The expression of human hARGHase gene product can also be analyzed by Northern blot technique, that is, the presence or absence and quantity of RNA transcript of human hARGHase in cells can be analyzed

Northern blot analysis of human hARGHase DNA and Western blot analysis of human hARGHase-specific antibodies can be used in combination to confirm the expression of human hARGHase in biological samples. Human hARGHase DNA can also be used for Southern blot analysis or in situ hybridization analysis to locate the gene on the chromosome, and genetic linkage analysis can be performed to find out other possible disease-related genes.

In addition, the present invention also provides a nucleic acid molecule that can be used as a probe, which generally has 8-100 consecutive nucleotides of the human hARGHase nucleotide coding sequence, preferably has 15-50 consecutive nucleotides acid. The probe can be used to detect whether there is a nucleic acid molecule encoding human hARGHase in a sample.

The present invention also provides a method for detecting whether there is a human hARGHase nucleotide sequence in a sample, which comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the human hARGHase nucleotide coding sequence and can be located on both sides or in the middle of the coding sequence. Primers are generally 15-50 nucleotides in length.

In addition, according to the human hARGHase nucleotide sequence and amino acid sequence of the present invention, human hARGHase homologous genes or homologous proteins can be screened on the basis of nucleic acid homology or expressed protein homology.

In order to obtain the dot array of human cDNAs or genomic DNAs related to the human hARGHase gene, the human cDNA or genomic DNA library can be screened with DNA probes that use ³² P to all or part of human hARGHase under low stringency conditions obtained by radioactive labeling. The most suitable cDNA library for screening is the library from human liver cancer tissue. cDNA libraries from other human tissues involved in endocrine or specific human cell lines may also be used for screening purposes. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are commercially available, for example, from Clontech, Palo Alto, Cal. This screening method can identify the nucleotide sequences of gene families related to human hARGHase.

Screening of human hARGHase homologues based on nucleotide similarity can be accomplished as follows. Human liver cancer tissue cDNA library, such as Clontech Cat.#74XX (Clontech, Palo Alto, Cal.) can be screened with a random primer DNA probe containing all or part of the human hARGHase gene sequence. To complete the identification of clones with DNA insertion sequences at least 70% homologous to the human hARGHase sequence, a hybridization solution with a hybridization temperature of 55° C. can be used, followed by washing with 0.5×SSC and 0.1% SDS. The cloned DNA insert identified in this way can be further evaluated for similarity to the human hARGHase gene by DNA restriction enzyme analysis and DNA sequencing. The distribution of tissue expression can be analyzed using the Northern blotting technique described above.

Human hARGHase homologs can also be identified with antibodies against human hARGHase protein or polypeptide. For example, commercially available or constructed expression libraries derived from cells or tissues such as liver cancer tissues can be screened by standard methods. The library is poured into a plate, and the colony is transferred to a nitrocellulose membrane, allowing the expressed recombinant protein to bind to the membrane. Typical antibody binding and detection can then be performed with a specific human hARGHase antibody. The DNA insert in the clone identified by this method can be further analyzed by DNA restriction enzyme analysis and DNA sequencing to evaluate its similarity to the human hARGHase gene. Tissue expression profiles of newly identified genes can be analyzed similarly as described above.

The full-length human hARGHase nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial chemical synthesis. Before this application, in the prior art, it was completely possible to obtain the nucleic acid sequence encoding the human hARGHase protein of the present invention by first synthesizing multiple small polynucleotide fragments and then connecting them. Then, the nucleic acid sequence can be introduced into various existing DNA molecules (such as vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the invention can also be produced by direct peptide synthesis using solid-phase techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

The coding sequence of the protein of the present invention can be used for gene mapping. For example, by fluorescent in situ hybridization (FISH), cDNA clones can be hybridized with metaphase chromosomes to accurately locate chromosomes. The technique can use cDNAs as short as about 500 bp; cDNAs as long as about 2000 bp or longer can also be used. For this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise location on a chromosome, it will be possible to correlate the sequence's physical location on the chromosome with genetic map data. Such genetic map data are available, for example, through the Mendelian Human Genetics Database (available online through the Johns Hopkins University Welch Medical Library). Linkage analysis is then used to identify associations between genes and diseases that have been mapped to the same chromosomal region.

Next, it is necessary to determine the differences in cDNA or genome sequence between diseased and healthy individuals. If a mutation is present in some or all affected individuals but not in normal individuals, the mutation may be the cause of the disease.

Using the human hARGHase protein of the present invention, through various conventional screening methods, substances that interact with human hARGHase, or receptors, inhibitors or antagonists, etc. can be screened out.

When the human hARGHase protein of the present invention and its antibody, inhibitor, antagonist, or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the human hARGHase protein of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of protein and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The human hARGHase protein of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When the human hARGHase protein polypeptide of the present invention is used as a medicine, a therapeutically effective dose of the polypeptide can be administered to mammals, wherein the therapeutically effective dose is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dosage is about 10 microgram/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Table 2 is the homology comparison (FASTA) table of the amino acid sequence (GenPept Accession No.AAC06773) of the human hARGHase protein of the present invention and the ARGHase protein of Aquifex aeolicus. Among them, the same amino acid is marked with a single amino acid character between the two sequences, and the similar amino acid is marked with "+".

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Example 1

Cloning of Human hARGHase Gene

1. Tissue separation (liver cancer tissue isolation)

Liver cancer tissues were derived from 5 normal adult male donors. The liver cancer tissues were taken out within four hours after death and immediately placed in liquid nitrogen for cryopreservation.

2. Isolation of mRNA (mRNA isolation)

Take out the tissue, grind it with a mortar, add it into a 50ml tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to a new 50ml tube and extract total RNA (TRIzol Reagents, Gibco, NY, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis. The mRNA in the total RNA was separated and quantified using a cellulose column with Oligo d(T).

3. Construction of cDNA library

Using mRNA as a template, double-stranded cDNA is synthesized, and the reverse transcription primer is shown in SEQ ID NO.1. After blunting the ends, add adapters containing EcoRI cutting points, and the adapter sequences are shown in SEQ ID NO.2 and 3, respectively. After phosphorylation of the EcoRI terminus, it was digested with XhoI restriction endonuclease for 1.5 hours before fragment isolation. Fragments with a length >500bp were screened through the column, extracted with phenol-chloroform, precipitated with ethanol, dissolved in sterile water, connected to the Uni-ZAP XR vector (Stratagene, CA9203, USA), and Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene , CA9203, USA) were packaged, and the host bacteria used XL 1-Blue MRF (Stratagene, CA9203, USA) bacteria. Plate and measure titer.

4. Sequencing and Database Constructing

The clones with foreign fragments inserted in the library were selected, and the plasmids were extracted after amplification (Qiagen, Germany). T3 and T7 were used as universal primers at the 3' and 5' ends, and terminators were fluorescently labeled (Big-Dye, Perkin- Elmer, USA) method, EST large-scale sequencing was carried out on ABI 377 sequencer (Perkin-Elmer, USA). The sequencing results were removed from the carrier sequence by FACTURA software and transferred to SUN Ultra 450 Server for further processing. All the sequence information is then searched in the existing database (Genebank+EMBL) with BLAST and FASTA software in the GCG software package (Wisconsin group, USA), and sequences with no homology or homology lower than 95% are regarded as new Gene database.

5. Full-length cloning of the gene (Cloning of Full-length cDNA)

On the basis of the obtained new gene fragment sequence information, the cDNA full-length clone is carried out in two stages:

(1) "Electronic Cloning"

Use the new gene fragment sequence as a probe to search the dbEST database, and consider the expressed sequence tag (Expressed Sequence Tag, “EST” for short) sequence with an overlapping sequence > 50bp and a homology of more than 98% as the same sequence (consensus sequence), take it out and use AUTOASSEMBLER software for splicing, part of EST can extend the probe sequence. Then use STRIDER software to analyze whether the extended sequence has a complete open reading frame (Open Reading Frame, ORF), and use BLAST to search Genbank or GenPept to determine whether the sequence has homology with other species at the nucleotide and amino acid levels , to help determine the full-length integrity of the obtained gene. By means of electronic cloning, the full-length sequence of human hARGHase gene can usually be obtained.

(2) Rapid Amplification of cDNA Ends (RACE)

If the complete cDNA full-length has not been obtained by the "electronic cloning" method, then design primers at the 5' or 3' end of the existing sequence, and carry out in the human liver cancer tissue Marathon-Ready cDNA library (Clontech Lab, Inc, USA) Long range PCR reaction. The PCR products were then cloned and sequenced. Use AUTOASSEMBLER and STRIDER software to analyze whether the extended sequence has a complete ORF, if not, repeat the above process until the full length is obtained.

(3) RT-PCR

For the known sequences at the 5' and 3' ends, if there is still a gap in the middle that cannot be obtained from the existing public database or its own database, RT-PCR may be considered. Primers were designed at the 5' end of the sequence, and Oligo-dT was used as a primer at the 3' end to amplify in the total RNA library of liver cancer tissues. The products were then cloned and sequenced. Finally splice and get the full length.

By using the above three methods in combination, the full-length coding sequence of the candidate human hARGHase protein was obtained. On the basis of splicing to obtain the full length (including at least the complete open reading frame), further design primer R1: 5'-TAGGGAAACTGTGAGGGCG-3' (SEQ ID NO.4) as the forward primer, oligonucleotide R2: 5' -CTGTCCCTTCATCCACACAAC-3'(SEQ ID NO.5) was used as a reverse primer, and the total RNA of liver cancer tissue was used as a template for RT-PCR amplification. The PCR conditions of R1/R2 were 94°C for 5 minutes, followed by 94°C 35 cycles of 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The PCR amplification product was detected by electrophoresis, and the length of the amplified fragment was 1582 bp. Then, the PCR amplification product was cloned and sequenced according to conventional methods to obtain the sequence shown in SEQ ID NO.6.

Example 2

Sequence information and homology analysis of human hARGHase gene: The length of the new full-length cDNA of human hARGHase of the present invention (GenBank Accession No.AF212236. It has not been disclosed before this application) is 1582 bp, and the detailed sequence is shown in SEQ ID NO.6 , wherein the open reading frame is located at 51-1142 nucleotides. The amino acid sequence of human hARGHase was deduced according to the full-length cDNA, with a total of 363 amino acid residues, a molecular weight of 40071.57, and a pI of 6.52. See SEQ ID NO.7 for the detailed sequence.

The full-length human hARGHase protein was searched for protein homology in the Non-redundant GenBank CDStranslations+PDB+GenPept+Superdate+PIR database using BLAST program, and it was found that it had certain homology with the ARGHase gene of Aquifex aeolicus. At the amino acid level, it has 30.0% identity and 59.6% similarity with the 18th-325th amino acid residues of the ARGHase protein of Aquifex aeolicus (GenPept Accession No. AAC06773) (see Table 2). It can be seen from the above that the human hARGHase gene and the ARGHase gene of Aquifex aeolicus have high homology at the protein level, belong to the same family and have high similarity in function.

In addition to being used as a member of the family for further functional research, the human hARGHase of the present invention can also be used to produce fusion proteins together with other proteins, such as producing fusion proteins together with immunoglobulins. In addition, the human hARGHase of the present invention can also be fused or exchanged fragments with other members of the family to produce new proteins. For example, the N-terminal of the human hARGHase protein of the present invention is exchanged with the N-terminal of the ARGHase protein of Aquifex aeolicus to produce new proteins with higher activity or new properties.

The antibody against human hARGHase of the present invention is used for screening other members of this family, or for affinity purification of related proteins (such as other members of this family).

In addition, the human hARGHase nucleic acid (coding sequence or antisense sequence) of the present invention can be introduced into cells to increase the expression level of human hARGHase or inhibit the overexpression of human hARGHase. The human hARGHase protein or its active polypeptide fragments of the present invention can be administered to patients to treat or alleviate related diseases caused by the lack, non-function or abnormality of human hARGHase. In addition, the nucleic acid sequence or antibody based on the present invention can also be used for relevant diagnosis or prognosis.

Since the human hARGHase protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or lower activity when administered to humans, compared to homologous proteins derived from other species (such as Aquifex aeolicus). Low side effects (eg, less or no immunogenicity in humans).

Example 3

Tissue expression profile of human hARGHase gene

Electronic Northern Expression Profiles. According to Ton C. et al.'s method (Ton C et al., Biochem BiophysRes Commun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation 1997 Dec 16; 96 (12): 4146-4203 ), the human hARGHase cDNA sequence was searched by BLAST in the dbEST database in the GCG software package, among the obtained human ESTs, there were hundreds of ESTs with probability values <10e-10 and identity >95%, which could be regarded as the The transcriptional expression of the gene in the tissue, from which the tissue profile of the gene expression is obtained, and it is found in colon cancer, neuroepithelial cells, parathyroid tumors, placenta, infant heart, T-cell lymphadenoma, testis, fetal brain , skeletal muscle and other organs or tissues, indicating that it plays a role in some important tissues or organs of the human body.

Example 4

Preparation and purification of human hARGHase polypeptide

In this example, the full-length human hARGHase coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

Prokaryotic expression of human hARGHase polypeptide in the form of GST fusion protein in Escherichia coli.

Construction of prokaryotic expression vector and transformation of Escherichia coli

According to the full-length coding sequence of human hARGHase (SEQ ID NO.6), primers were designed to amplify the complete coding reading frame (corresponding to about 20 or more nucleotides at the 5' and 3' ends of the coding sequence respectively), and Restriction endonuclease sites were introduced into the anti-primers (this depends on the pGEX-2T vector selected), so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the human hARGHase gene was cloned into the pGEX-2T vector (Pharmacia, Piscataway, NJ) under the premise of ensuring the correct reading frame. The identified expression vector was transformed into Escherichia coli DH5α by the CaCl ₂ method, and the engineering strain DH5α-pGEX-2T-hARGHase containing the pGEX-2T-hARGHase expression vector was screened and identified.

Isolation and Identification of Engineering Bacteria Expressing GST-hARGHase Recombinant Protein

Pick the DH5α-pGEX-2T-hARGHase engineered bacterium of a single colony and shake it in 3ml LB medium containing 100μg/ml ampicillin for overnight culture, draw the culture solution into the new LB medium (containing 100μg /ml ampicillin) for about 3 hours, and when the OD ₆₀₀ reached 0.5, IPTG was added to a final concentration of 1 mmol/L to continue culturing at 37°C for 0, 1, 2, and 3 hours, respectively. Centrifuge 1ml of bacterial solution with different culture time, add lysate (50μl of 2×SDS loading buffer, 45μl of distilled water, 5μl of dimercaptoethanol) to the bacterial sediment, suspend the bacterial sediment, boil in boiling water bath for 5 minutes, 10000rpm Centrifuge for 1 minute, add the supernatant to 12% SDS-PAGE gel electrophoresis. After staining, it was observed that the amount of protein with expected molecular weight increased with the increase of IPTG induction time, which was the engineering bacteria expressing GST-hARGHase fusion protein.

Extraction and Purification of GST-hARGHase Fusion Protein

The engineered bacteria DH5α-pGEX-2T-hARGHase expressing GST-hARGHase fusion expression protein was induced according to the above method. The induced bacteria were centrifuged and precipitated, and 20ml of PBS was added to resuspend the bacteria for every 400ml of bacteria, and the bacteria were sonicated. Add 50% glutathione Sepharose 4B saturated with PBS in an amount of 20 microliters per milliliter to the ultrasonic solution that has completely broken the bacteria, shake at 37°C for 30 minutes, and centrifuge at 10,000 rpm for 10 minutes to precipitate glutathione bound to GST-hARGHase Glycoside peptide Sepharose 4B, discard the supernatant. Add 100 μl of PBS to the precipitate obtained in each milliliter of ultrasonic liquid to wash twice, then add 10 μl of reduced glutathione eluent to the precipitate obtained in each milliliter of ultrasonic liquid, place at room temperature for 10 minutes, centrifuge at 10,000 rpm for 10 minutes, and the supernatant is Eluted fusion protein. Repeat the elution twice. The eluted supernatant was stored at -80°C, and subjected to SDS-PAGE electrophoresis to detect the purification effect. The protein band at 40kDa is human hARGHase protein.

Example 5

Eukaryotic expression of human hARGHase protein or polypeptide in insect cells

1. Construction of human hARGHase baculovirus expression vector and transfection to Sf9 insect cell line

According to the full-length coding sequence of human hARGHase (SEQ ID NO.6), design primers to amplify the complete coding reading frame, and introduce restriction endonuclease sites on the forward and reverse primers respectively (this may vary depending on the carrier selected). ) to construct expression vectors. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the human hARGHase cDNA was cloned into pVL1392 vector (Invitrogen, Carlsbad, CA) under the premise of ensuring the reading frame. Add 3 μg of the identified expression vector, 1 μg of wild-type linear baculovirus DNA (BaculoGold ^TM ACMNPV DNA, Pharmingen, San Diego, CA) and 25 μl of Lipofection (Gibco-BRL, NY) to 1 ml of serum-free insect medium, shake Mix for 15 seconds and incubate at room temperature for 15 minutes. Take 1ml (2×10 ⁶ ) of Sf9 insect cell suspension in a 60mm tissue culture plate, change the transfection medium after 1 hour of attachment, discard the medium after incubating at room temperature for 15 minutes, and add the prepared DNA vector transfection mixture , Seal the culture plate with Parafilm, shake and culture at room temperature 27°C for 4 hours, then change the complete medium and culture for 3 days, and collect the supernatant for use.

2. Screening and identification of insect cell lines transformed into recombinant expression vectors

Insect cells 3 days after transfection were made into cell suspension (2×10 ⁶ /1ml) with fresh medium, 1ml of cell suspension was placed in a 60mm tissue culture dish, 3ml of medium was added, and 100μl of culture supernatant collected, Adhere to the wall for 1 hour, discard 2ml of medium, continue to culture at room temperature for 1 hour, discard all the medium, add 3ml of preheated semi-solid medium containing 20μl of 4% X-gal, pick white cell clones after 5-7 days of culture Cultured in a 96-well culture plate for 3-5 days, and then aspirate the supernatant to infect Sf9 insect cells.

Infected cells were collected for Western identification. After the cells were lysed, SDS-PAGE electrophoresis was performed. The gel after electrophoresis was transferred to the nitrocellulose membrane in Pharmacia's Multiphor II semi-dry electrotransfer instrument, and the nitrocellulose membrane was placed in the blocking solution for blocking for 1 hour, and then in Block in anti-human hARGHase antibody solution for 1 hour, shake and wash with TBS solution for 5 minutes, a total of 2 times, then place the membrane in biotin-labeled anti-human hARGHase primary antibody secondary antibody solution, shake for 1 hour, wash with TBS, Add avidin-alkaline phosphatase complex to react for 30 minutes, wash twice with TBS, add freshly prepared chromogenic solution to develop color and observe protein bands.

Sf9 cell clones that highly express human hARGHase were picked.

3. Extraction and purification of human hARGHase protein

The supernatant of Sf9 cell clones highly expressing human hARGHase was used to infect Sf9 cells in large quantities. After 48 hours of infection, the cells were collected and washed with PBS. Add 20ml of cell lysate (0.5% Triton X-100, 20mM Na ₃ PO ₄ (sodium phosphate, pH7.8), 500mM NaCl, 1mM Na ₃ VO ₄ (sodium vanadate), 1mM Pefabloc, for every 2×10 ⁸ cells 1 μg/ml pepsin, leupeptin and aprotinin) to break the cells, centrifuge at 12000×g for 20min to remove cell debris, add 2ml NTA-agarose (Qiagen, Germany) to the supernatant for every 2× ¹⁰⁸ cells , adsorption at 4°C for 1 hour. Then, it was washed twice with His buffer containing 100 nM imidazole, and eluted with a buffer containing 20 mM N,N'-dipiperazine, 500 mM NaCl, and 300 mM imidazole to obtain a purified protein. The eluate was stored at 4°C, and the purity of the extracted human hARGHase protein was detected by SDS-PAGE electrophoresis. The protein band at 40kDa is human hARGHase protein.

Example 6

Preparation of anti-human hARGHase antibody

1. Preparation of ARGHase and splenocytes of immunized mice: the human hARGHase protein obtained in Example 5 and Example 6 is separated by chromatography and then used for later use, or can be separated by SDS-PAGE gel electrophoresis. Electrophoretic bands were excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Take the ARGHase of 6-8 weeks old Balb/C female mice, and inject 50-100 μg/0.2ml of emulsified protein intraperitoneally to the ARGHase of the mice. After 14 days, the same antigen emulsified with incomplete Freund's adjuvant was used to boost the ARGHase of the mouse once more at a dose of 50-100 μg/0.2ml, and it was used for fusion after 3-5 days. Among them, for the preparation of spleen cells, see E Zheng, editor-in-chief, "Tissue Culture and Molecular Cytology", Beijing Publishing House, p. 210.

2. Prepare feeder cells according to the method in "Tissue Culture and Molecular Cytology Techniques" (supra), p. 371.

3. Carry out cell fusion according to the method in "Tissue Culture and Molecular Cytology Techniques" (supra), p. 213.

4. Antibody detection: After 10-15 days of cell fusion, it is necessary to check well by well. Once vigorous hybrid cell colony growth is found, human hARGHase protein is used for preliminary screening of antibody activity. Commonly used methods include: immunofluorescence test, Emission immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). Immediately after the wells with antibody activity were detected, the clones were cultured and the antibodies were isolated.

The sequences and symbols involved in the present invention are listed as follows:

(1) Information on SEQ ID NO.1

(i) Sequence features:

(A) Length: 50bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(ix) Sequence description: SEQ ID NO.1GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT 50

(2) Information of SEQ ID NO.2

(i) Sequence features:

(A) Length: 13bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(ix) Sequence description: SEQ ID NO.2

AATTCGGCAC GA G 13

(3) Information of SEQ ID NO.3

(i) Sequence features:

(A) Length: 9bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(ix) Sequence description: SEQ ID NO.3

GCCGTGCTC 9

(4) Information of SEQ ID NO.4

(i). Sequence features:

(A) Length: 19bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii).Molecular type: oligonucleotide

(ix). Sequence description: SEQ ID NO.4TAGGGAAACTGTGAGGGCG 19

(5) Information on SEQ ID NO.5

(i). Sequence features:

(A) Length: 21bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii). Molecular type: oligonucleotide (ix). Sequence description: SEQ ID NO.5CTGTCCCTTTCATCCACACAAC 21

(6) Information of SEQ ID NO.6

(i) Sequence features:

(A) Length: 1582bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: Nucleotide (ix) Sequence description: SEQ ID NO.6

1 TAGGGAAACT GTGAGGGCGG CACCGGAAGT GGCGAGCAGT CTGCGCGCGG

51 ATGGCCCAGC GGCGATGGCG GCACGGCAGG TTGGAGGGGC TGGCGCGGCC

101 GCTCCCTCTC GCGCTTCCGA GGCTGCCTGG CTGGCGCGCT GCTCGGGGAC

151 TGCGTGGGCT CCTTCTACGA GGCCCACGAC ACCGTCGACC TGACGTCATC

201 GTGCGTCATG TCCAGAGTCT GGAGCCGGAC CCCGTACTGC CCCGGGAAGT

251 GAGCGGACAG AAGCCTTTTGT ACTACACAGA TGACACAGCC ATGGCCAGGG

301 CCCTGGTGCA GTCCCTGCTA GCCAAGGAGG CCTTTGACGA GGTGGACATG

351 GCTCACAGAT TTGCTCAGGA GTACAAGAAA GACCCTGACA GGGGCTATGG

401 TGCTGGAGTA GTCACTGTCT TCAAGAAGCT CCTGAACCCC AAATGTCGCG

451 ATGTCTTTGA GCCTGCCCGG GCCCAGTTTA ACGGGAAAGG CTCCTATGGC

501 AATGGAGGTG CCATGCGGGT GGCTGGCATC TCCCTGGCCT ATAGCAGTGT

551 CCAGGATGTG CAGAAGTTTG CCCGGCTCTC GGCCCAGCTG ACACACGCCT

601 CCTCCCTGGG TTACAATGGC GCCATCCTGC AGGCCCTGGC TGTGCACCTG

651 GCCTTGCAGG GCGAGTCTTC CAGCGAGCAC TTTCTCAAGC AACTCCTGGG

701 CCACATGGAG GATCTGGAGG GTGATGCCCA GTCCGTCTTG GATGCCAGGG

751 AGTTGGGCAT GGAGGAGCGT CCATACTCCA GCCGCCTGAA GAAGATTGGA

801 GAGCTTCTAG ACCAGGCATC GGTGACCAGG GAGGAAGTGG TGTCTGAGCT

851 AGGGAATGGC ATTGCTGCCT TTGAGTCGGT ACCCACCGCC ATCTACTGCT

901 TCCTACGCTG CATGGAGCCA GACCCTGAGA TCCCTTCTGC CTTCAATAGC

951 CTCCAAAGGA CTCTCATTTA TTCCATCTCA CTTGGTGGGG ACACAGACAC

1001 CATTGCCACC ATGGCTGGGG CCATTGCTGG TGCCTACTAT GGGATGGATC

1051 AGGTGCCAGA GAGCTGGCAG CAAAGCTGTG AAGGCTACGA GGAGACAGAC

1101 ATCCTGGCCC AAAGCCTGCA CCGTGTCTTC CAGAAGAGTT GATGAGGGCT

1151 ACAGCTGTTG GGGCTCTGCA GGTCCCCTGG GACACACTAC AGCTCCAATC

1201 AGAAACCCTG CGCTTCCTTG AGTGTGGCTT CCCACTTTTC CTGCATTGTG

1251 GAGCTGACTG AGTACACCGG TGAGGCTGGG GTCTCTGCAG GGGAGGTCAC

1301 TGGAACAGCG AGCAAGGGAC TGGTGCCTCG CTGGTGCTGG GTCTCTGGTT

1351 TGCTGCAGAG CCGTAGGACA CTCCTGGCTC CTCAGTAGGA CAGACAGACG

1401 CAGGCGGGTT TATTTTGGAG GGGTACTTGT GGCATTTTCC TGTATTGTCT

1451 TGGACATGGG ATGTGGGGAG GTGGAAATGA TGAGCAGTAG CATCATTTCT

1501 CCCTGTTGGG TTTTAGCCAG TTTGCCAGCA AGCGCATCCT AGCAGGGTCC

1551 CCGAGCAGCA GGTTGTGTGG ATGAAGGGAC AG

(7) Information of SEQ ID NO.7

(i) Sequence features:

(A) Length: 363 amino acids

(B) type: amino acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: polypeptide

(ix) Sequence description: SEQ ID NO.7:

1 MAQRRWRHGR LEGLARPLPL ALPRLPGWRA ARGLRGLLLR GPRHRRPDVI

51 VRHVQSLEPD PVLPREVSGQ KPLYYTDDTA MARALVQSLL AKEAFDEVDM

101 AHRFAQEYKK DPDRGYGAGV VTVFKKLLNP KCRDVFEPAR AQFNGKGSYG

151 NGGAMRVAGI SLAYSSVQDV QKFARLSAQL THASSLGYNG AILQALAVHL

201 ALQGESSSEH FLKQLLGHME DLEGDAQSVL DARELGMEER PYSSRLKKIG

251 ELLDQASVTR EEVVSELGNG IAAFESVPTA IYCFLRCMEP DPEIPSAFNS

301 LQRTLIYSIS LGGDTDTIAT MAGAIAGAYY GMDQVPESWQ QSCEGYEETD

351 ILAQSLHRVF QKS table 230.0% identity in 307 aa overlap, 59.6% similarity in 307 aa overlap

30 40 50 60 70 80h.pep RAARGLRGLLLRGPRHRRPDVIVRHVQSLEPDPVLPREVSGQKPLYYTDDTAMARALVQS

| | | | + || | +|+|++ | |++|a.pep VGDALGKSVEDITEEEVFEFYGDRIRDFVTPHPSSP--AYGQLPEETSDETTIFRRLLES

20 30 40 50 60 70

90 100 110 120 130 140h.pep LLAKEAFDEVDMAHRFAQEYKKDPDRGYGAGVVTVFKKLLNPKCRDVFEPARAQFNGKGS

|+ |+|+| |+ +|+ + | ++ + | ++ + ||+ | + +|+ +| |a.pep LVEKKALDVRDFLNRLMEWYDREEMHRYPDPMLLTAIDLLS---RGI-NPST---HGLTS

80 90 100 110 120

                                                                     

| +| ++++| | + + + + +++ + ||| |+ ++ +||| || +|| ++|+a.pep FSVEGILRSVALGLYHYYNPELAAEGSKVVSLLTHRSDVISDASAILGALIAHL-VRGDF

130 140 150 160 170 180

210 220 230 240 250 260h.pep SSEHFLKQLLGHMEDLEGDAQSVLDARELGMEERPYSSRLKKIGELLDQASVTREEVVSE

| | ++ | +| |+ |+ ||+ +++ | +++||| + |+ | +++a.pep YLEDF-REKLRLIETLKDYAK----------EEK-HKKILDRVAELL -MESADLE TAINT

190 200 210 220 230

                                                               

|||| |||+ | |+| || +| |+ + +| || + +||||+|+ ++||+a.pep LGNGTFAFEAFPLALYIFLSNVENPEEALFQAVNSYGE-------FGGDTDAIGFLVGAM

240 250 260 270 280

330 340 350 360h.pep AGAYYGMDQVPESWQQSCEGYEETDILAQSLHRVFQKS

||||| + +| +++ |+ ++ ||+ |++| +|+a.pep LGAYYGEEAIPYHLRENVENSQKLRELAERLYEVVEKQVGFSQ

290 300 310 320 330h.pep: protein sequence of human hARGHase a.pep: protein sequence of Aquifex aeolicus ARGHase (GenPept Accession No.AAC06773)

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hARGHase protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 51-1142 position among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 51-1142 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 51-1142 position.

4. isolated people hARGHase protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6, it is characterized in that it comprises prokaryotic cell prokaryocyte and eukaryotic cell.

8. a generation has the method for the polypeptide of people hARGHase protein active, is characterised in that its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of people hARGHase protein-active operationally is connected in expression regulation sequence, form people hARGHase protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 51-1142 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of people hARGHase;

(3) under the condition that is fit to expressing human hARGHase protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with people hARGHase protein-active.

9. energy and the described people hARGHase of claim 7 protein polypeptide specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.

10. a nucleic acid molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

11. one kind is used for the method whether test sample exists people hARGHase nucleotide sequence, it is characterized in that it comprises with described probe of claim 10 and sample hybridizes, whether detection probes combination has taken place then, this sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to people hARGHase nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, primer length is 15～50 Nucleotide.